Lecithin cholesteryl acyltransferase (LCAT) is a 63 kD enzyme that is responsible for the esterification of virtually all free cholesterol in plasma and thus, is essential for the process of reverse cholesterol transport. Cholesteryl esters formed by LCAT are preferentially incorporated into HDL particles. Patients with functional defects in LCAT can present with remarkably different clinical features, based on either a partial enzyme deficiency, designated Fish Eye Disease (FED) or a total enzyme deficiency, designated Classical LCAT Deficiency (CLD). In our studies we have investigated the underlying molecular defects in the LCAT gene that lead to the expression of these two phenotypically distinct clinical syndromes. We have identified a novel mutation that leads to FED in two siblings of French descent presenting with corneal opacifications and HLD-cholesterol levels of less than 10 mg/dl. DNA sequencing and RFLP analysis revealed that the patients were true homozygotes for a triplet deletion resulting in the loss of leu-300. In vitro expression and characterization of the variant demonstrated that the mutant enzyme had a specific alpha-LCAT activity similar to normal LCAT but accumulated intracellularly. These studies indicate that deletion of leu-300 results in a partial LCAT secretion defect leading to the expression of FED in this kindred. To further investigate the role that different LCAT gene mutations play in the expression of FED or CLD, 8 different mutant LCAT were expressed in human embryonal kidney-293 cells and characterized by using a proteoliposome activity assay. The alpha-LCAT specific activity was reduced to less than 5% of control in 6 FED and CLD mutants alike. However, CLD mutant 156 and FED mutant 300 preserved 30% and 100% of control alpha-LCAT specific activity. Both of these LCAT variants accumulated intracellularly. Thus, in contrast to previous data, total absence of alpha-LCAT activity is not a pre-requisite for development of FED. These studies establish the heterogeneity of molecular defects that can result in either FED or CLD.